A peer-reviewed open-access journal ZooKeys 277: 91–108 (2013) The Italian alien molluscan state of knowledge updated 91 doi: 10.3897/zookeys.277.4362 RESEARCH ARTICLE www.zookeys.org Launched to accelerate biodiversity research Alien molluscan species established along the Italian shores: an update, with discussions on some Mediterranean “alien species” categories Fabio Crocetta1, Armando Macali2, Giulia Furfaro2, Samantha Cooke3, Guido Villani4, Ángel Valdés3 1 Stazione Zoologica Anton Dohrn, Villa Comunale, I-80121 Napoli, Italy 2 Dipartimento di Biologia, Uni- versità Roma Tre, Viale Marconi 446, I-00146 Roma, Italy 3 Department of Biological Sciences, California State Polytechnic University, 3801 West Temple Avenue, Pomona, California 91768-4032, USA 4 Istituto di Chimica Biomolecolare (CNR), Via Campi Flegrei 34, I-80078 Pozzuoli (Napoli), Italy Corresponding author: Fabio Crocetta ([email protected]) Academic editor: N. Yonow | Received 19 November 2012 | Accepted 6 March 2013 | Published 15 March 2013 Citation: Crocetta F, Macali A, Furfaro F, Cooke S, Villani G, Valdés Á (2013) Alien molluscan species established along the Italian shores: an update, with discussions on some Mediterranean “alien species” categories. ZooKeys 277: 91–108. doi: 10.3897/zookeys.277.4362 Abstract The state of knowledge of the alien marine Mollusca in Italy is reviewed and updated. Littorina saxatilis (Olivi, 1792), Polycera hedgpethi Er. Marcus, 1964 and Haminoea japonica Pilsbry, 1895 are here considered as established on the basis of published and unpublished data, and recent records of the latter considerably expand its known Mediterranean range to the Tyrrhenian Sea. COI sequences obtained indicate that a comprehensive survey of additional European localities is needed to elucidate the dispersal pathways of H. japonica. Recent records and interpretation of several molluscan taxa as alien are discussed both in light of new Mediterranean (published and unpublished) records and of four categories previously excluded from alien species lists. Within this framework, ten taxa are no longer considered as alien species, or their records from Italy are refuted. Furthermore, Trochocochlea castriotae Bellini, 1903 is considered a new synonym for Gibbula albida (Gmelin, 1791). Data provided here leave unchanged as 35 the number of alien molluscan taxa recorded from Italy as well as the percentage of the most plausible vectors of introduction, but raise to 22 the number of established species along the Italian shores during the 2005–2010 period, and backdate to 1792 the first introduction of an alien molluscan species (L. saxatilis) to the Italian shores. Keywords Alien Mollusca, natural entries, translocations, state of knowledge, Italy Copyright Fabio Crocetta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 92 Fabio Crocetta et al. / ZooKeys 277: 91–108 (2013) Introduction Although marine invasions have been well documented all over the world, they are particularly conspicuous in the Mediterranean Sea, mainly due to the high number of vectors of introduction (Galil 2009, 2012; Zenetos et al. 2010, 2012). Moreover, the complex geological history of the region, and the fact that the basin was almost entirely re-colonized naturally by the Atlantic Ocean fauna (Harzhauser et al. 2007) - and this natural interference is still in progress - makes the situation in the Mediterranean even more complex, with the possibility that relatively recent natural dispersals can be inter- preted as human-mediated introductions. In this paper we examine some molluscan species from the Mediterranean Sea that appear to be the result of human introductions, with special emphasis on Italy. The previously published state of knowledge of Italian alien molluscan species for the period 2005-2010 (see Crocetta 2011, 2012) is updated based on both molecular data and faunal observations. Additionally, discussions of sev- eral taxa belonging to four categories previously excluded by Crocetta (2012) from alien species lists [1- species recorded on the basis of empty shells only; 2- possible crypto- genic or vagrant species (here listed as “Deep-water species, natural dispersers or spe- cies with a plausible Atlanto-Mediterranean distribution”); 3- species with a complex/ unclear taxonomy; 4- translocations of native Mediterranean species to an area where they previously did not occur], but recently recorded from Italy or included among “Mediterranean aliens” by other authors, are relevant to the entire Mediterranean basin. Materials and methods Alien species definition and establishment status The inclusion (or not) of the taxa listed as aliens in the Mediterranean is based on strict accordance to the definition of alien species by the International Union for Conserva- tion of Nature: ‘[non-native, non-indigenous, foreign, exotic] means a species, subspecies, or lower taxon occurring outside of its natural range (past or present) and dispersal potential (i.e., outside the range it occupies naturally or could not occupy without direct or indirect introduction or care by humans) and includes any part, gametes or propagule of such species that might survive and subsequently reproduce.’ Four categories previously excluded (Crocetta 2012), but included by other au- thors, are discussed. Additionally, an alien species is considered as “established” if, dur- ing the surveyed period, at least one self-maintaining population is currently known to occur in the wild (see Crocetta 2012). The definition of cryptogenic species follows Carlton (1996): ‘a species that cannot be included with confidence among native nor among introduced ones.’ The Italian alien molluscan state of knowledge updated 93 Taxonomy, nomenclature, published and unpublished data collection A survey of published Mediterranean records of the taxa listed in the present paper was conducted and, where necessary, a full list of synonyms for Mediterranean records is provided. Unpublished records, when available, are listed under each species and come from the examination of preserved specimens, photographs and personal observations obtained from different research projects conducted in several sites along the Italian coasts. Updated taxonomy and nomenclature used follow WoRMS (World Register of Marine Species: last accessed 30 January 2013), unless clearly specified [see Gibbula albida (Gmelin, 1791) for the new synonymy proposed]. DNA extraction, PCR sequencing and data analysis A total of 8 specimens of Haminoea japonica Pilsbry, 1895 from two populations in Italy (Lago Fusaro and Lago di Sabaudia) were sequenced for the cytochrome c oxi- dase I (COI) mitochondrial gene, following methods used in Hanson et al. (2013). Specimens were preserved in 99.8% ethyl alcohol. DNA was extracted from a small foot sample using a hot Chelex® protocol, then amplified by polymerase chain reac- tion (PCR) using the HCO2198/LCO1490 universal primers for COI (Folmer et al. 1994). PCRs were performed in a 50 μL reaction volume containing 0.25 μL 5U/ μL taq polymerase, 5.00 μL 10x buffer, 5.00 μL 25 mM MgCl2, 1.00 μL 40 mM dNTPs, 1.00 μL each 10 mM primer, 34.75 μL H2O, and 2.00 μL extracted DNA. Reaction conditions involved an initial denaturation of 95°C for 3 min, 35 cycles of 94°C for 45 s, 45°C for 45 s, and 72°C for 2 min, followed by a final elongation step of 72°C for 10 min. PCR products were run on gel electrophoresis to confirm the presence of DNA fragments of appropriate size (700 bp), and positive products were cleaned using Montage PCR Cleanup Kit (Millipore). The DNA concentra- tion of purified samples was then determined using a NanoDrop 1000 spectropho- tometer (Thermo Scientific). Sequencing was outsourced to the City of Hope DNA Sequencing Laboratory (Duarte, California, USA) using sequencing buffer BigDye V3.1. Sequences were assembled and edited using the software Geneious pro 4.7.4 (Biomatters Ltd.). Geneious also was used to extract the consensus sequences and to align the sequences using the default parameters. In order to determine the relation- ships between the haplotypes found in Lago Fusaro and Lago di Sabaudia and other haplotypes found in the non-native range of H. japonica, a haplotype network was constructed using TCS 1.21 (Clement et al. 2000) with a fixed connection limit of 100 steps. Sequence data have been submitted to the GenBank databases with acces- sion numbers JX679598-JX679605. Data for non-Italian haplotypes were obtained from Hanson et al. (2013). 94 Fabio Crocetta et al. / ZooKeys 277: 91–108 (2013) Results and discussion Alien molluscan species established along the Italian shores In order to verify the presence or absence of alien species, a continuous monitoring effort is necessary. Only field surveys may provide such evidence, and the new records of the two species reported below ensure that they fit wel into the category of established species. Family LITTORINIDAE Children, 1834 Littorina saxatilis (Olivi, 1792) Unpublished material examined. Italy - Veneto: Malamocco (45°20.35'N, 12°18.80'E), under and on rocks and in crevices at tidal level. Voucher specimens: 05/2009, approx. 50 sps., legit F. Crocetta and F. Favero; 06/2009, approx. 100 sps., legit F. Crocetta and F. Favero; 03/2010, approx. 20 sps., legit F. Crocetta. Remarks. Littorina saxatilis (Olivi, 1792) was originally